This invention relates to polypeptides forming antigen binding structures with specificity for Rhesus D antigens and especially to Fab molecules with specificity for the Rhesus D antigen. The invention also relates to their application to provide pharmacological and diagnostic compositions. The above Fab fragments when genetically engineered to be part of complete antibodies are useful for the prophylaxis of hemolytic disease of the newborn (HDN). This invention provides the novel DNA and amino acid sequences of the above polypeptides.
Thus, the antibodies can be used for the protection of Rhesus negative women before or immediately after the birth of a Rhesus positive child to prevent HDN in subsequent pregnancies.
The invention also includes the application of the said Fab molecules either alone or in combination with Fc constant regions as complete antibodies for the purposes of treating other illnesses which might benefit from anti-Rhesus D immunoglobulin e.g. treatment of idiopathic thrombocytopenic purpura (ITP).
In addition anti-Rhesus D immunoglobulin can be used after mistransfusions of Rhesus positive blood to Rhesus negative recipients in order to prevent sensitization to the Rhesus D antigen. Further the invention relates to the application of these Fab fragments and antibodies as diagnostic reagents.
HDN is the general designation for hemolytic anemia of fetuses and newborn babies caused by antibodies of the mother. These antibodies are directed against antigens on the surface of the fetal erythrocytes. These antigens can belong to the Rhesus, ABO or other blood group systems.
The Rhesus blood group system includes 5 major antigens: D, C, c, E and e (Issitt, P. D., Med. Lab. Sci. 45:395, 1988). The D antigen is the most important of these antigens as it is highly immunogenic eliciting anti-Rhesus D antibodies during Rhesus incompatible pregnancies and following transfusion of Rhesus incompatible blood. The D antigen is found in approximately 85% of Caucasians in Europe and those individuals are said to be Rhesus positive, Individuals lacking the D antigen are called Rhesus negative. The expression of the D antigen can vary due to either low antigen density, hereafter known as weak D or Du, or due to partial antigenicity, hereafter known as partial D antigens.
The Rhesus D antigen, a membrane protein of the erythrocyte, has recently been cloned and its primary structure described (Le Van Kim, C., et al., PNAS 89:10925, 1992). Modeling studies suggest that the Rhesus D antigen has 12 transmembrane domains with only very short connecting regions extending outside the cell membrane or protruding into the cytoplasm.
The partial D phenotypes were first identified in people who carried D antigen on their red cells but who had an alloanti-D in their sera (Rose, R. R. and Sanger, R., Blood groups in man, Blackwell Scientific, Oxford, U.K. 1975; Tippett, P. et al., Vox Sanguinis. 70:123, 1996). This can be explained by regarding the D antigen as a mosaic structure with at least 9 different epitopes (epD1 to epD9). Thus in some D variant people the red cells lack part of this mosaic and antibodies are made to the missing D epitopes. Rhesus positive individuals that make antibodies against partial D antigens have been classified into six main different categories (DII to DVII) each having a different abnormality in the D antigen. More recently it has been shown that these D categories gave different patterns of reaction when tested against panels of human monoclonal anti-D antibodies (Tippett, P., et al., Vox Sanguinis. 70:123, 1996). The different reaction patterns identified the 9 epitopes and so define the different partial D categories. The number of epitopes present on the D antigen varies from one partial D category to another with the DVI category expressing the least, epD3, 4 and 9. The DVI category is clinically important as a DVI woman can be immunized strongly enough to cause hemolytic disease of the newborn.
The prophylactic efficacy of anti-RhD IgG for prevention of hemolytic disease of the newborn is well established and has been in routine use for many years. As a result this severe disease has become a rarity. Nevertheless the underlying cause of the disease, i.e. RhD incompatibility between a RhD negative mother carrying a RhD positive child still remains and thus requires a continual supply of therapeutic anti-RhD IgG.
In recent years the assurance of a continual supply of anti-RhD IgG has become an increasing problem. The pool of available hyperimmune serum from alloimmunized multiparous Rhesus negative women has drastically decreased due to the success of prophylactic anti-RhD. Thus the current methods of production require repeated immunization of an increasingly reluctant pool of donors for the production of high titer antiserum (Selinger, M., Br. J. Obstet. Gynaecol. 98:509, 1991). There are also associated risk factors and technical problems such as the use of Rhesus positive red blood cells for repeated immunization carrying the risk of transmission of viral diseases like hepatitis B, AIDS and other as yet unknown viruses (Hughes-Jones, N. C., Br. J. Haematol. 70:263, 1988). Therefore an alternative method for production of anti-RhD antibodies is required.
In the past few years various alternative sources of hyperimmune serum have been tried but all are associated with disadvantages. Epstein Barr Virus (EBV) transformation of lymphocytes creating B lymphoblastoid cell lines that secrete specific antibody including against the Rhesus D antigen (Crawford et al., Lancet. 386: Feb. 19th, 1983) are unstable and require extensive cloning. Also due to the low transformation efficiencies (1-3% of B cells) only a restricted range of antibody specificities can be obtained from the potential repertoire. Additionally it seems that mice do not respond to the Rhesus D antigen and thus no murine monoclonal antibodies are available which could be used for producing chimaeric or humanised antibodies. Until recently the only other alternative was production of human antibodies by the hybridoma technique which was also restricted by the lack of a suitable human myeloma cell fusion partner (Kozbor, D. and Roder, J. C., Immunol. Today. 4:72, 1983).